This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.107.105338v1 93/6/2143    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Smith, M. I. Articles by Roberts, C. J. Search for Related Content PubMed PubMed Citation Articles by Smith, M. I. Articles by Roberts, C. J.

Nucleation and Growth of Insulin Fibrils in Bulk Solution and at Hydrophobic Polystyrene Surfaces

M. I. Smith ^*, J. S. Sharp ^* and C. J. Roberts 

^* School of Physics and Astronomy and Nottingham Nanotechnology and Nanoscience Centre, and Laboratory of Biophysics and Surface Analysis, School of Pharmacy, and Nottingham Nanotechnology and Nanoscience Centre, The University of Nottingham, Nottingham, NG7 2RD, United Kingdom

Correspondence: Address reprint requests to J. S. Sharp, School of Physics and Astronomy and Nottingham Nanotechnology and Nanoscience Centre, The University of Nottingham, Nottingham, NG7 2RD, UK. E-mail: james.sharp{at}nottingham.ac.uk.

A technique was developed for studying the nucleation and growth of fibrillar protein aggregates. Fourier transform infrared and attenuated total reflection spectroscopy were used to measure changes in the intermolecular ß-sheet content of bovine pancreatic insulin in bulk solution and on model polystyrene (PS) surfaces at pH 1. The kinetics of ß-sheet formation were shown to evolve in two stages. Combined Fourier transform infrared, dynamic light scattering, atomic force microscopy, and thioflavin-T fluorescence measurements confirmed that the first stage in the kinetics was related to the formation of nonfibrillar aggregates that have a radius of 13 ± 1 nm. The second stage was found to be associated with the growth of insulin fibrils. The ß-sheet kinetics in this second stage were used to determine the nucleation and growth rates of fibrils over a range of temperatures between 60°C and 80°C. The nucleation and growth rates were shown to display Arrhenius kinetics, and the associated energy barriers were extracted for fibrils formed in bulk solution and at PS surfaces. These experiments showed that fibrils are nucleated more quickly in the presence of hydrophobic PS surfaces but that the corresponding fibril growth rates decrease. These observations are interpreted in terms of the differences in the attempt frequencies and energy barriers associated with the nucleation and growth of fibrils. They are also discussed in the context of differences in protein concentration, mobility, and conformational and colloidal stability that exist between insulin molecules in bulk solution and those that are localized at hydrophobic PS interfaces.

This article has been cited by other articles:

				M. I. Smith, J. S. Sharp, and C. J. Roberts Insulin Fibril Nucleation: The Role of Prefibrillar Aggregates Biophys. J., October 1, 2008; 95(7): 3400 - 3406. [Abstract] [Full Text] [PDF]

				V. Smirnovas and R. Winter Revealing Different Aggregation Pathways of Amyloidogenic Proteins by Ultrasound Velocimetry Biophys. J., April 15, 2008; 94(8): 3241 - 3246. [Abstract] [Full Text] [PDF]